Australian Centre for Blood Diseases, Monash University, Melbourne, Victoria, Australia.
J Thromb Haemost. 2012 Jun;10(6):1133-41. doi: 10.1111/j.1538-7836.2012.04734.x.
Ligation of the platelet-specific collagen receptor, GPVI/FcRγ, causes rapid, transient disulfide-dependent homodimerization, and the production of intracellular reactive oxygen species (ROS) generated by the NADPH oxidase, linked to GPVI via TRAF4.
The aim of this study was to evaluate the role of early signaling events in ROS generation following engagement of either GPVI/FcRγ or a second immunoreceptor tyrosine-based activation motif (ITAM)-containing receptor on platelets, FcγRIIa.
Using an H(2) DCF-DA-based flow cytometric assay to measure intracellular ROS, we show that treatment of platelets with either the GPVI agonists, collagen-related peptide (CRP) or convulxin (Cvx), or the FcγRIIa agonist 14A2, increased intraplatelet ROS; other platelet agonists such as ADP and TRAP did not. Basal ROS in platelet-rich plasma from 14 healthy donors displayed little inter-individual variability. CRP, Cvx or 14A2 induced an initial burst of ROS within 2 min followed by additional ROS reaching a plateau after 15-20 min. The Syk inhibitor BAY61-3606, which blocks ITAM-dependent signaling, had no effect on the initial ROS burst, but completely inhibited the second phase.
Together, these results show for the first time that ROS generation downstream of GPVI or FcγRIIa consists of two distinct phases: an initial Syk-independent burst followed by additional Syk-dependent generation.
血小板特异性胶原受体 GPVI/FcRγ 的交联会导致快速、短暂的二硫键依赖同源二聚化，以及 NADPH 氧化酶产生的细胞内活性氧物质 (ROS)，通过 TRAF4 与 GPVI 相连。
本研究旨在评估在血小板上交联 GPVI/FcRγ 或第二个包含免疫受体酪氨酸激活基序 (ITAM) 的受体 FcγRIIa 后，ROS 生成的早期信号事件的作用。
使用基于 H(2) DCF-DA 的流式细胞术测定来测量细胞内 ROS，我们表明，用 GPVI 激动剂胶原相关肽 (CRP) 或 convulxin (Cvx) 或 FcγRIIa 激动剂 14A2 处理血小板会增加血小板内 ROS；其他血小板激动剂如 ADP 和 TRAP 则不会。来自 14 位健康供体的富含血小板的血浆中的基础 ROS 显示出个体间的差异很小。CRP、Cvx 或 14A2 在 2 分钟内诱导初始 ROS 爆发，随后在 15-20 分钟后达到平台期的额外 ROS。抑制 ITAM 依赖性信号的 Syk 抑制剂 BAY61-3606 对初始 ROS 爆发没有影响，但完全抑制了第二阶段。
这些结果首次表明，GPVI 或 FcγRIIa 下游的 ROS 生成由两个不同的阶段组成：最初的 Syk 非依赖性爆发，随后是额外的 Syk 依赖性生成。
